High throughput oil-emulsion synthesis of bowtie barcodes for paired mrna capture and sequencing from individual cells

ABSTRACT

Methods for incorporation of unique bowtie-barcodes into a nucleic acid origami nanostructure (FIG. 1). In particular, provided herein are methods that facilitate pairing and analysis of nucleic acids from individual cells using, for example, high-throughput next-generation sequencing.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from and benefit of U.S. ProvisionalApplication No. 62/377,123, filed on Aug. 19, 2016, the disclosure ofwhich is incorporated by reference herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under R21 CA196460 andR21 A112587 awarded by the National institutes of Health. The governmenthas certain rights in the invention.

BACKGROUND

There are many biological questions that require single-cell analysis ofmultiple gene sequences, including analysis of clonally distributeddimeric immunoreceptors on lymphocytes and the accumulation ofdriver/accessory mutations in polyclonal tumors. Lysis of bulk cellpopulations results in the mixing of gene sequences, making itimpossible to know which pairs of gene sequences originated from anyparticular cell and obfuscating analysis of rare sequences within largecell populations. Although current single-cell sorting technologies canbe used to address some of these questions, such approaches areexpensive, require specialized equipment, and lack the necessaryhigh-throughput capacity for comprehensive analysis of large samples.Accordingly, there remains a need in the art for improved methods forgenetic analysis of individual cells.

BRIEF SUMMARY

In a first aspect, provided herein is a method for incorporatingsingle-stranded DNA (ssDNA) barcoded polynucleotides into a nucleic acidorigami nanostructure. The method can comprise or consist essentially of(a) performing first strand synthesis of a barcode nucleic acid togenerate double-stranded barcode nucleic acids, wherein the barcodenucleic acid comprises at least one priming site complementary to oneside of a single-stranded 5′-5′ bowtie linker nucleic acid comprising acentral 5′-5′ phosphodiester linker flanked on either side bycomplementary barcode nucleic acids and sequence(s) complementary to anucleic acid origami nanostructure that may be conducted prior to orduring subsequent oil-emulsion amplification steps; (b) combining thedouble-stranded barcode nucleic acids (or the single-stranded barcodeand complementary primer pairs) and the 5′-5′ bowtie linker nucleic acidin an oil-emulsion droplet comprising reagents for amplifying a targetnucleic acid; (c) thermal cycling the oil-emulsion droplet comprisingthe double-stranded (or single-stranded) barcode nucleic acids primersand the 5′-5′ bowtie linker nucleic acid sufficient to result inannealing of each strand from the double-stranded barcode nucleic acidsto complementary sequences on the 5′-5′ bowtie linker nucleic acid andelongation of ssDNA barcoded 5′-5′ bowtie polynucleotides therebyyielding a product including barcoded mRNA capture sequences on eitherside of the 5′5′ phosphodiester linkage bowtie strand; (d) extractingelongation products from the thermal cycled droplet; (e) purifying ssDNAbarcoded 5′-5′ bowtie polynucleotides from the extracted elongationproducts; and (f) incorporating the purified ssDNA barcoded 5′-5′ bowtiepolynucleotides into a nucleic acid origami nanostructure. Incorporatingcan comprise annealing the purified ssDNA barcoded 5′-5′ bowtiepolynucleotides to a nucleic acid origami nanostructure. The nucleicacid origami nanostructure can be a DNA nanostructure.

In another aspect, provided herein is a method for detecting targetnucleic acid sequences at the single cell level. The method can compriseor consist essentially of (a) contacting a nucleic acid origaminanostructure obtained according to the method of claim 1 to nucleicacids isolated from a single cell, wherein the nanostructure comprisesssDNA barcoded polynucleotides having barcoded sequences complementaryto target nucleic acid sequences; and wherein contacting occurs underconditions suitable for binding of the barcoded sequences to the targetnucleic acid sequence if present in the single cell; (b) recoveringtarget nucleic acid sequences bound to the barcoded sequences; and (c)reverse transcribing the recovered target nucleic acid sequences usingthe ssDNA barcoded polynucleotides as gene-specific primers for reversetranscription, whereby target nucleic acids, if present in the cell, aredetected without a single-cell sorting step. The nucleic acid origaminanostructure can be a DNA nanostructure.

In a further aspect, provided herein is a method for detecting B cellreceptor sequences at the single cell level. The method can comprise orconsist essentially of (a) transfecting into an antigen-specific B cellexpressing unique heavy (IgH) and light (IgL) chain BCR mRNA; (b)contact a DNA origami nanostructure comprising multiple barcoded mRNAcapture sequences to capture and protect both immunoglobulin heavy (IgH)and light (IgL) chain BCR mRNA in transfected antigen-specific B cells;(c) isolating the contacted DNA origami nanostructures to recover IgHand IgL mRNA bound to the barcoded mRNA capture sequences; and (d)reverse transcribing the recovered IgH and IgL mRNA using the barcodedmRNA capture probes as gene-specific primers for reverse transcription,whereby target BCR sequences are detected without a single-cell sortingstep. The DNA origami nanostructures can comprise integral biotin labelsand wherein isolating the contacted nanostructures comprises avidincolumn purification.

The foregoing and other advantages of the invention will appear from thefollowing description. In the description, reference is made to theaccompanying drawings, which form a part hereof, and in which there isshown by way of illustration a preferred embodiment of the invention.Such embodiment does not necessarily represent the full scope of theinvention, however, and reference is made therefore to the claims andherein for interpreting the scope of the invention.

BRIEF DESCRIPTION OF DRAWINGS

The present invention will be better understood and features, aspects,and advantages other than those set forth above will become apparentwhen consideration is given to the following detailed descriptionthereof. Such detailed description makes reference to the followingdrawings, wherein:

FIG. 1 is a schematic of an exemplary method of using DNA origaminanostructures to obtain linked sequence information from BCR/antibodymRNAs from single cells without single-cell sorting. Antigen-reactive Bcells are obtained by flow cytometry cell sorting using fluorescentlylabeled antigens and transfected by electroporation with DNA origaminanostructures containing extended sequences complementary to both IgHand IgL (i.e, κ or λ) constant region mRNA. DNA origami molecules bindand protect intracellular IgH and IgL mRNA within individual cells thatare then lysed, and origami with bound mRNA are then reisolated andpurified. Using the mRNA capture probes as reverse transcriptionprimers, IgH and IgL gene sequences are extended on the origami captureprobes. The cDNA is then further amplified by standard IgH/IgL V-genemultiplex PCR to obtain a pool of amplification products suitable forIllumina paired-end sequencing. Each amplicon contains a 12-mer barcodethat can be paired to its complement, thus providing sequenceinformation for both IgH and IgL mRNA from an individual cell withoutthe need for single-cell sorting.

FIGS. 2A-2C illustrate DNA origami design and synthesis. (A)Organization of 5′-5′ bowtie mRNA capture probe. Various regions allowfor incorporation into DNA origami nanostructures, downstream PCRamplification, barcode pairing, and IgH/IgL mRNA capture. (B) Schematicvisualization of 5′-5′ bowtie mRNA capture probe extending from thesurface of an origami nanostructure while annealing with BCR IgH and IgLmRNA (note: the length of the 5′-5′ bowtie capture probes has beenexaggerated to allow for visualization of the structure). (C) Validationof properly folded DNA origami molecules as visualized by AFM showingthe anticipated “wafer” shape (probes are too flexible to be visualizedby AFM). Each origami molecule is roughly 60×90 nM in scale.

FIGS. 3A-3C illustrate construction of 5′-5′ bowtie mRNA capture probescontaining complementary barcode sequences. (A) Following first strandssynthesis of 10-mer barcode strands, a 1:1:1 barcode:5′-5′strand:oil-water emulsion droplet reaction is set up. (B) Overlapextension of the 5′-5′ strands using the barcode strands as templatesallows for complementary barcodes to be incorporated on either end ofthe 5′-5′ strands. (C) Following denaturing PAGE purification, adapterstrands are utilized to perform a modular T4 DNA ligation reaction, toattach gene-specific complementary mRNA capture sequences to either endof the 5′-5′ strand. After a final denaturing PAGE purification andquantification, the barcoded 5′-5′ mRNA capture probes can beincorporated into individual DNA origami nanostructures.

FIGS. 4A-4C demonstrate binding of DNA origami nanostructures to targetmRNA. (A) Target mRNA binds specifically to the DNA origami structures:Shift of DNA origami due to specific binding of target mRNA wasvisualized using a reporter fluorescein molecule. Lanes 1-3: DNA origamiwithout target-specific extended staple sequences incubated with invitro transcribed target mRNA (1), alone (2), or with nonsense(scrambled) mRNA (3). Lane 4: In vitro transcribed target mRNA only (notvisible), Lane 5: MW marker (not visible), Lanes 6-7: DNA origami withextended target-specific staple sequences incubated alone (6) or with invitro transcribed target mRNA (7). The specific FRET signal identifiesDNA origami structures. (B) Selected AFM images show specificity of invitro transcribed target mRNA bound to origami with target-specificextended staples but not to non-target staples (mRNA visible only on oneside of the origami molecule). (C) Selected AFM images show binding oftwo in vitro transcribed target mRNAs, captured by individual origamicontaining both alpha and beta staples (mRNA bound on both sides oforigami molecules).

FIGS. 5A-5B demonstrate transfection of primary lymphocytes with DNAorigami nanostructures. (A) Lymphocytes from mice were mock transfected(left) or transfected with FITC-labeled DNA origami nanostructures(middle). Transfected cells were visualized based on detection of FITClabel by flow cytometry. To ensure that origami structures were beingtaken up by cells and not bound to the cell surface, cells were treatedwith DNase after transfection. A similar FITC signal was detected fromthe DNA origami following DNase treatment (right). (B) DNase digestiondestroys DNA origami on the cell surface after incubation, as confirmedby gel electrophoresis.

FIG. 6 is a flowchart illustrating an exemplary Next GenerationSequencing (NGS) analysis protocol. Sequences from individual runs areparsed into FASTQ files for alignment using BWA-MEM software. Analysisfiles are generated for mapping, read count, and junction detection, andfurther processed for quality control of sequences and amplificationbias.

While the present invention is susceptible to various modifications andalternative forms, exemplary embodiments thereof are shown by way ofexample in the drawings and are herein described in detail. It should beunderstood, however, that the description of exemplary embodiments isnot intended to limit the invention to the particular forms disclosed,but on the contrary, the intention is to cover all modifications,equivalents and alternatives falling within the spirit and scope of theinvention as defined by the appended claims.

DETAILED DESCRIPTION

All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as though set forth in their entirety in the presentapplication.

The inventors previously developed novel DNA origami nanostructureshaving the capacity to capture and protect mRNA sequences fromtransfected cells. Integral fluorescent labels on the DNA origamifacilitate identification of transfected cells, and reisolation of thenanostructures with bound mRNA is achieved using integral biotin labelsand avidin column purification. The methods provided herein are based atleast in part on the inventors' discovery of a robust methodology forpairing multiple genes of interest from individual cells without theneed for single cell sorting or specialized equipment (FIG. 1). Thepresent disclosure provides novel methods for incorporating“bowtie-barcode” technology into DNA origami nanostructures and othernucleic acid-based nanostructures to allow for simultaneous pairing andcapture of mRNAs from millions of cells. Additional improvements inmicroporation (using microliter volumes for cell electroporation)technology have increased transfection efficiency and improved cellviability. Finally, the combination of the bowtie-barcode capturemethods with oil-water emulsion droplet based systems and overlapextension PCR enables the creation of millions of paired, randomsequences that can be utilized as barcodes to link individual geneticsequences back to one another during sequencing. By linking mRNAsthrough unique matching barcoded sequence analysis, the inventorsdeveloped a cost-effective method useful for analyzing large cellpopulations and easily adaptable for probing sequence diversity ofpaired genes from virtually any heterogeneous cell population.

With respect to other transfectable nucleic acid hybridizationplatforms, no other successful molecular approaches exist for ananalysis requiring the linking of multiple mRNA species. Whiletransfection of single stranded oligos has been attempted, the singlestranded oligos activate the RISC complex and are degraded, precludingdownstream analysis. Without being bound to any particular mechanism ortheory, it is believed that the semi-supercoiled nature of the origamimolecules inhibits degradation by the RISC complex, as evidenced by theability to re-capture and isolate transfected origami molecules withbound mRNAs. Additionally, in reference to standard single-celledemulsion-based systems, they were not suitable for analysis requiringthe linking of multiple mRNA species. While the emulsion-based systemallows for input of higher cell numbers than single cell sorting, priorart single-celled emulsion-based systems only captured 7 pairedsequences (>0.000001%) and were unable to detect the presence of theircontrol at a concentration less than 1000/1000000. While most clones arebelieved to exist as a single clone per person, this level ofsensitivity would not be acceptable for discovery of rare cells.

Accordingly, in a first aspect, provided herein is a method ofincorporating single-stranded DNA (ssDNA) barcoded polynucleotides intoa nucleic acid origami nanostructure. The method comprises, or consistsessentially of, the following steps: (a) performing first strandsynthesis of a barcode nucleic acid (either prior to or duringsubsequent oil-emulsion steps) to generate complementary double-strandedbarcode nucleic acids, where the barcode nucleic acid comprises at leastone priming site complementary to one portion of a single-strandedbowtie linker DNA strand comprising a central 5′-5′ phosphodiesterlinker flanked by sequences complementary to the barcode nucleic acidsand sequence(s) complementary to a nucleic acid origami nanostructure;(b) combining the double-stranded barcode nucleic acids (orsingle-stranded barcode nucleic acids and complementary priming sets)and the 5′-5′ bowtie linker nucleic acid in an oil-emulsion dropletcomprising reagents for elongating a target nucleic acid; (c) thermalcycling the oil-emulsion droplet comprising the double-stranded barcodenucleic acids (or single-stranded barcode nucleic acids andcomplementary primers) and the 5′-5′ bowtie linker nucleic acidsufficient to result in annealing of each strand from thedouble-stranded barcode nucleic acids to complementary sequences on the5′-5′ bowtie linker nucleic acid and elongation of single stranded DNA(ssDNA) barcoded 5′-5′ bowtie polynucleotides thereby yielding anelongation product droplet; (d) extracting elongation products from thedroplet; (e) purifying ssDNA barcoded 5′-5′ bowtie polynucleotides fromthe extracted elongation products; and (f) incorporating the purifiedssDNA barcoded 5′-5′ bowtie polynucleotides into a nucleic acid origaminanostructure.

As used herein, the term “barcode” refers to unique sequences ofnucleotides that can be used to distinguish, pair, and uniquely identifynucleic acids from the same cell. In some cases, barcodes may be used todistinguish tens, hundreds, or even thousands of nucleic acids, e.g.,arising from different cells or other sources. As used herein, the term“bowtie-barcode” refers to a set of complementary barcode sequenceslinked together via a unique 5′-5′ non-standard phosphodiester DNA“bowtie” structure, meaning a central phosphodiester linker flanked oneither side by complementary barcode nucleic acid sequences.

As used herein, the term “nucleic acid nanostructure” is used forconvenience and it is to be understood that the invention contemplatesnucleic acid nanostructures generally. The nanostructures of theinvention may be linear (e.g., nanorods) or non-linear (e.g.,star-shaped, triangular, etc.). Nucleic acids such as DNA or RNA may befolded into predetermined one-, two- or three-dimensional nanostructuresusing a variety of techniques, such as DNA or RNA origami.

The terms “oligonucleotide,” “nucleotide,” and “nucleic acid” are usedinterchangeably to mean molecules comprising a sugar (e.g., ribose ordeoxyribose) linked to a phosphate group and to an exchangeable organicbase, which is either a substituted pyrimidine (e.g., cytosine (C),thymidine (T) or uracil (U)) or a substituted purine (e.g., adenine (A)or guanine (G)). Thus, the term embraces both DNA and RNAoligonucleotides. The terms shall also include polynucleosides (i.e., apolynucleotide minus the phosphate) and any other organicbase-containing polymer. Oligonucleotides can be obtained from existingnucleic acid sources (e.g., genomic or cDNA), but are preferablysynthetic (e.g., produced by nucleic acid synthesis).

Construction of barcoded 5′-5′ bowtie linker capture probes: Barcodedpolynucleotides are long ssDNA strands, constructed containing a central5′-5′ “bowtie” linkage, allowing for both ends to run 5′→3′. The bowtielinkages can comprise a pre-synthesized DNA sequence (e.g., ordered fromcommercial vendor). Preferably, either end of a barcoded polynucleotideadditionally may comprise a specific sequence complementary to theM13mp18 phage DNA origami backbone. This sequence can be varied andadapted for barcode incorporation into any target nanostructure.Referring to FIG. 2A, either end can additionally comprise a conservedPCR primer site for downstream amplification, a unique barcodecomplementary to the barcode on the opposing end of the strand, a secondconserved priming site, and an mRNA capture site complementary to aconserved region of the genes of interest. An important design featureof the capture sequences is that mRNAs are paired to one another by aunique set of complementary nucleotide barcodes contained on either endof the 5′-5′ bowtie strand.

The long barcoded bowtie polynucleotide can then be incorporated intothe DNA origami mastermix (or other desired nanostructure) and theM13mp18 complementary sequence(s) in the barcode bowtie self-assemblewith the origami nanostructure. Referring to FIG. 2B, the mRNA capturesequence regions of the bowtie extend from the surface of the origaminanostructure. In some cases, incorporating a ssDNA 5′-5′ barcodedbowtie polynucleotide into a nucleic acid nanostructure can compriseannealing.

As described herein, oil-emulsion droplets can be used to incorporate aunique set of complementary barcodes into millions (or more) bowtiestrands. Base bowtie strands comprising central 5′-5′ non-traditionalphosphodiester linkages can include sequence(s) complementary tosequences of a nucleic acid origami nanostructure as well as twodifferent conserved priming sites, which are procured from a commercialvendor (e.g., Integrated DNA Technologies, Inc.). In some cases, the5′-5′ bowtie strand has, for example, the following sequence:

(SEQ ID NO: 1) 3′-CGAGTCCCTTTATCGGGAAC-5′-5′-GAACGTGGCGAGAAAGGAAGGGAACAAACTATGGACAGCAAAGACAGCACCT-3′.

In some cases, the barcode strand has the following sequence:

(SEQ ID NO: 2) 5′CACCGACTTTGACTCCCAAATCAATGTGCGGACAGCAAAGACAGCACCTNNNNNNNNNNNNGCTCAGGGAAATAGCCCTTGGGGTAGCCT TTTGTTTGTTTGCAATCTCTG3′,where “N” can be any of the four nitrogen bases found in DNA (adenine,cytosine, guanine, or thymine).

In other cases, the barcode labeled strands comprise one priming sitethat is complementary to one side of a 5′-5′ bowtie strand; a random10-mer nucleotide barcode (4¹⁰=1048576 unique barcodes, if >10⁶sequences are analyzed an 11-mer or 12-mer barcode may be employed); anda second priming site that is complementary to the other side of the5′-5′ bowtie strand with specific mRNA (complementary) capture sequencesflanking both ends of the barcode strand. Short barcode strands may beused in a first-strand-synthesis reaction to create dsDNA products withcomplementary barcodes or used downstream in standard polymerase chainreaction (PCR) reactions during the oil-emulsion elongation step tosynthesize the dsDNA barcode strands directly in the emulsion droplet.Referring to FIG. 3A, >10¹⁰ identical ssDNA 5′-5′ bowtie strands andunique dsDNA barcode strands (or ssDNA barcode strands withcomplementary primers) are then incorporated at a 1:1:1molecule-to-molecule-to-droplet ratio in an oil-water emulsion dropletoverlap extension elongation system. Standard PCR cycles can be used todenature the dsDNA barcode strands (or amplify the ssDNA barcode strandsfollowed by denaturation), to anneal both ssDNA strands from the dsDNAbarcode strands with either end of a 5′-5′ bowtie, and to elongate thebowtie using the ssDNA barcode strands as a template.

After denaturation, overlap extension is carried out in each droplet aseach of the strands from the dsDNA barcode will anneal with itscomplementary priming site on the 5′-5′ bowtie linker, acting as aprimer and template for overlap extension, and thus incorporatingcomplementary 10-mer barcodes on either end of the 5′-5′ bowtie strandsas well as mRNA specific capture sequences on the 3′ ends of the bowtiestrands (FIG. 3B). While this system is not an exponential PCRamplification, multiple dissociation/annealing/elongation cycles areemployed to ensure both ends of each 5′-5′ linker are elongated. PCRproducts are then extracted from the oil-water emulsion system using astandard ether/ethyl acetate extraction protocol, and ssDNA barcoded5′-5′ bowtie strands are purified from remaining nucleic acids bystandard denaturing PAGE purification. Products at this point willcontain (in order from 5′-5′ central bowtie linkage extending towardsboth 3′ ends) the following: origami complementary sequence (may be oneside only), a conserved PCR priming site, a random 10-mer barcodecomplementary to the barcode on the opposing arm of the 5′-5′ linkage,and a second conserved priming site, and gene specific mRNA capturesequences (FIG. 3B). Following the final denaturing PAGE purification,final products will contain regions necessary for: 1) incorporation intothe DNA origami structure, 2) conserved priming sites for downstream PCRamplification, 3) complementary barcodes utilized for pairing bothcaptured mRNAs, and 4) mRNA capture sequences complementary to conservedregions of genes of interest that can also be used as gene-specificreverse transcription primers (FIG. 3C).

These unique bowtie barcodes can then be incorporated into individualorigami molecules or any nucleic acid structure of interest by simplychanging the identity of the “origami annealing sequence” portion of thestrand. This method allows for extreme levels of mRNA pairingspecificity due to the uniquely barcoded mRNA capture sequences attachedto each bowtie strand.

The recovered mRNAs are reverse transcribed (RT) into cDNA usingconserved sequences on the capture strands as gene-specific RT primers,thereby extending the gene-specific cDNA from the 3′ ends of thebowtie-barcodes. Since the conserved gene-specific sequences on thebowtie barcodes are required for priming the RT reaction, any unboundmRNAs will not be amplified, thus improving selectivity and avoidingfalse pairing.

Reverse transcription and barcode-linked mRNA amplicon generation (FIG.1): Once mRNA-bound origami has been purified from cell lysate, reversetranscription (RT) will be performed. Using the mRNA complementaryregions of the bowtie barcodes as RT primers, elongation can be achievedby simply adding any commercially available RT mastertnix, andincubating per manufacturer's recommendations. The mRNA can then beremoved by addition of a commercial RNaseH cocktail, and barcoded cDNAwill be obtained as extended products from the 3′ ends of the barcodebowties. We have previously validated this approach using in vitrotranscribedtarget mRNA-bound origami and confirmed the ability toisolate gene specific cDNA by PCR. Finally, a multiplex PCR using asingle primer for both of the conserved priming sequences on either endof the bowtie barcodes, and, depending on the application of thetechnology, 1) the well-established multiplex primer sets for theTCRα/TCRβ V-genes or IgH/IgL V-families can be perfonned to generate apool of amplicons with corresponding CDR3 regions of immunoreceptors or2) a single primer for a conserved region just outside of the mutationof interest in known cancer driver/co-driver genes, will result inbarcode-paired PCR products. By inclusion of Illumina-specific adaptersequences in the primer sets, barcoded amplicons with CDR3 sequences,cancer mutation sequences, or any paired gene sequences of interest fromheterologous cell populations can be obtained that are immediatelysuitable for use in standard Illumina paired-end sequencing.

In another aspect, provided herein are methods for the parallel capture,barcoding, and quantification of a panel of tens to hundreds, or more,of specific DNA and/or RNA sequences from large numbers of single cells,e.g., for the purpose of profiling cell populations or other purposes.In preferred embodiments, a method for detecting target nucleic acidsequences at the single cell level comprises, or consists essentiallyof, the following steps: (a) contacting a nucleic acid origaminanostructure obtained according to the method of claim 1 to nucleicacids isolated from a single cell, wherein the nanostructure comprisesssDNA barcoded polynucleotides having barcode sequences complementary totarget nucleic acid sequences; and wherein contacting occurs underconditions suitable for binding of the barcode sequences to the targetnucleic acid sequence if present in the single cell; (b) recoveringtarget nucleic acid sequences bound to the barcode sequences; and (c)reverse transcribing the recovered target nucleic acid sequences usingthe ssDNA barcoded polynucleotides as gene-specific primers for reversetranscription, whereby target nucleic acids, if present in the cell, aredetected without a single-cell sorting step.

In a further aspect, provided herein is a method for detecting B cellreceptor sequences at the single cell level. The method comprises, orconsists essentially of, the following steps: (a) transfecting into anantigen-specific B cell; (b) contact a DNA origami nanostructurecomprising one or more sets of mRNA capture sequences to capture andprotect both immunoglobulin heavy (IgH) and light (IgL) chain BCR mRNAin transfected antigen-specific B cells (c) isolating the contacted DNAorigami nanostructures to recover IgH and IgL mRNA bound to the one ormore mRNA capture sequences; and (d) reverse transcribing the recoveredIgH and IgL mRNA using the one or more sets of mRNA capture probes asgene-specific primers for reverse transcription, whereby target BCRsequences are detected without a single-cell sorting step.

In some cases, the DNA origami nanostructures comprise integral biotinlabels, and the step of isolating the contacted nanostructures cancomprise avidin column purification.

A nucleic acid or nucleic acid molecule, as used herein, can include anynucleic acid of interest. In some embodiments, nucleic acids include,but are not limited to, DNA, RNA, peptide nucleic acid, morpholinonucleic acid, locked nucleic acid, glycol nucleic acid, threose nucleicacid, mixtures thereof, and hybrids thereof, or nucleic acids withinternal carbon backbone spacers. In some aspects, a nucleic acid is a“primer” capable of acting as a point of initiation of synthesis along acomplementary strand of nucleic acid when conditions are suitable forsynthesis of a primer extension product. Nucleic acids may besingle-stranded, double-stranded, and also tripled-stranded. In someaspects, the nucleic acid serves as a template for synthesis of acomplementary nucleic acid, e.g., by base-complementary incorporation ofnucleotide units. For example, in some aspects, a nucleic acid comprisesnaturally occurring DNA (including genomic DNA), RNA (including mRNA),and/or comprises a synthetic molecule including, but not limited to,complementary DNA (cDNA) and recombinant molecules generated in anymanner. In some aspects, the nucleic acid is generated from chemicalsynthesis, reverse transcription, DNA replication or a combination ofthese generating methods.

Nucleic acids can be obtained using any suitable method, including thosedescribed by Maniatis et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor, N.Y., pp. 280-281 (1982). In some aspects, nucleicacids are obtained as described in U.S. Patent Application PublicationNo. US2002/0190663. Nucleic acids obtained from biological samplestypically are fragmented to produce suitable fragments for analysis.

Nucleic acids and/or other moieties of the invention may be isolated. Asused herein, “isolated” means separate from at least some of thecomponents with which it is usually associated whether it is derivedfrom a naturally occurring source or made synthetically, in whole or inpart. Nucleic acids and/or other moieties of the invention may bepurified. As used herein, “purified” means separate from the majority ofother compounds or entities. A compound or moiety may be partiallypurified or substantially purified. Purity may be denoted by a weight byweight measure and may be determined using a variety of analyticaltechniques such as but not limited to mass spectrometry, HPLC, etc.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which the invention pertains. All definitions, as defined andused herein, should be understood to control over dictionarydefinitions, definitions in documents incorporated by reference, and/orordinary meanings of the defined terms.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one,”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e., “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein, the terms “approximately” or “about” in reference to anumber are generally taken to include numbers that fall within a rangeof 5% in either direction (greater than or less than) the number unlessotherwise stated or otherwise evident from the context (except wheresuch number would exceed 100% of a possible value). Where ranges arestated, the endpoints are included within the range unless otherwisestated or otherwise evident from the context.

The present invention has been described in terms of one or morepreferred embodiments, and it should be appreciated that manyequivalents, alternatives, variations, and modifications, aside fromthose expressly stated, are possible and within the scope of theinvention. The invention will be more fully understood uponconsideration of the following non-limiting Examples.

EXAMPLES Example 1—Reverse Transcription and Barcode-Linked mRNAAmplicon Generation

Once mRNA-bound origami has been purified from cell lysate, reversetranscription (RT) is performed. Using the mRNA complementary regions ofthe bowtie barcodes as RT primers, elongation is achieved by adding anycommercially available RT mastermix, and incubating per themanufacturer's recommendations. The mRNA is removed by addition of acommercial RNaseH cocktail, and barcoded cDNA is obtained as extendedproducts from the 3′ ends of the barcode bowties. We previouslyvalidated this approach using in vitro transcribedtarget mRNA-boundorigami and confirmed the ability to isolate gene specific cDNA by PCR.Finally, multiplex PCR is performed using a single primer for each ofthe conserved priming sequences on either end of the bowtie barcodes.Depending on the application of the technology, (1) the well-establishedmultiplex primer sets for the TCRα/TCRβ genes or BCR IgH/IgL V-familiesare to generate a pool of amplicons with corresponding CDR3 regions ofimmunoreceptors; or (2) a single primer for a conserved region justoutside of the mutation of interest in known cancer driver/co-drivergenes is used to obtain barcode-paired PCR products; or (3) a singleprimer for a conserved region adjacent to heterologous genes withindiverse organismal species or cell populations. By inclusion ofIllumina-specific adapter sequences in the primer sets, barcodedamplicons with CDR3 sequences are obtained that are immediately suitablefor use in standard Illumina paired-end sequencing.

Example 2—DNA Origami Binding to Immunoreceptor mRNA

We previously developed the following approach for analysis ofheterodimeric T cell receptors (TCR), which pose similar problems interms of diversity and heterogeneous cell populations. FIGS. 4A-4Cpresent preliminary data in the context of analysis of a knowntransgenic TCR (P14 transgenic T cells). Origami nanostructures weredesigned to comprise multiple mRNA capture probes per individual origamimolecule. We have since modified our nanostructure design to incorporatea single 5′-5′ barcoded mRNA capture strand per origami molecule. Whilethe mRNA binding kinetics are assumed to be similar, optimizationexperiments will be performed to evaluate mRNA binding efficiency.Previously, when origami containing both TCRα- and TCRβ-specific probeswere incubated with TCRβ mRNA only, mRNA was only captured by one sideof the origami molecule (presumably with TCRβ-specific probes) (FIG.4B). When origami containing both TCRα- and TCRβ-specific probes wasincubated with both TCRα and TCRβ mRNA, both sides of the origamicaptured mRNA indicating both types of TCR mRNA were bound tocorresponding probes on either side of individual origami molecules(FIG. 4C), confirming the capability and specificity of correct mRNAbinding. Again, for our prior experiments analyzing TCR mRNA binding, weutilized individual probe strands on either side of the origamimolecule, complementary for either TCRα or TCRβ sequences respectively.Although capture of mRNA will occur in a slightly different orientation,no significant change in origami-mRNA capture is expected.

Example 3—Transfection of DNA Origami Nanostructures

Previous studies have utilized receptor-mediated endocytosis of DNAnanostructures via the caveolin-dependent pathway leading to microtubuletransport to lysosomes and therefore breakdown of the nanostructure. Theobvious problem with this strategy is that for mRNA capture andsubsequent reisolation, our nanostructures must bypass the endocytosispathway, and instead cross the membrane directly to the cytoplasm.Electroporation is the simplest method for cytoplasmic entry and thusavoidance of the deleterious effects of endosomal degradation. Howeverhigh cell mortality rates have commonly been associated with thismethodology. Recent advancements in microporation technology (the use ofμL volume electroporations) have shown high transfection efficiencies(>93%) as well as high cell viabilities (>86%). For our B celltransfection experiments we will use KL25 IgL/IgH transgenic miceexpressing the same KL25 sequence BCR as the hybridoma that werepreviously generated [34]. Splenocytes from 4-to 6-week-old KL25 IgL/IgHtransgenic mice will be prepared by mechanical disruption and red bloodcell lysis in 0.83% NH₄Cl. IgM+B cells will be purified by magnetic cellsorting and >95% purity of sorted populations will be confirmed by flowcytometry. Cells will be pelleted by centrifugation (1200 rpm, 5minutes, 4° C.), washed with OPTI-MEM™ media (Gibco), and resuspended inOPTI-MEM™ media at 1×10⁷ cells/mL. For electroporation, a Neon syringetransfection system (Thermo Scientific) will be used with 100 μL syringetips at the following settings: 2000 V, 10 ms, 1 pulse. Samples willconsist of 100 μL cell suspensions and either 25 μL (20 nM) DNA origamisuspension (in 1X TAE-Mg²⁻) or a mock transfection control of 25 μL 1XTAE-Mg²⁻buffer. Immediately following electroporation, cells will betransferred to fresh RPMI-1640 culture medium with 10% fetal calf serumand incubated at 37° C. for 12-24 hours in individual wells of a 96 wellplate. To assess transfection efficiency, cells will be visualized on aLSR Fortessa flow cytometer; the fluorescein isothiocyanate (FITC; 488nm excitation, 518 nm emission) tag incorporated into each nanostructureallows for successffilly transfected cells to be identified by flowcytometry.

We previously developed this transfection protocol for mouse primary Tlymphocytes (FIG. 5A) and anticipate that similar settings will provideinitial starting points for B cell transfection. In our previousexperiments, we verified that the DNA origami nanostructures entertransfected lymphocytes rather than binding non-specifically to the cellsurface that would give false-positive readings by treating transfectedcells with concentrated DNase (Turbo DNase, Ambion, Life Technologies)and then analyzed the cells for successful transfection by FACS analysis(FIG. 5A). We also confirmed that concentrated DNase treatment resultedin the successful destruction of DNA nanostructures (FIG. 5B). Thus, DNAorigami has excellent transfection efficiency for primary lymphocytes.It should be noted that Turbo DNase is a proprietarily engineeredversion of DNaseI and has a markedly higher affinity for DNA than wildtype DNaseI, which we found to be much less effective at degradation oforigami nanostructures. Additionally, our group has previouslydemonstrated that DNA origami are highly stable in cell Lysate for 12hours [33], confirming these structures are resistant to cytosolicnuclease degradation.

Example 4—Sequence Data Management, Processing and Analysis (Prophetic)

The success of this project depends on a validated data processingpipeline and an experienced informatics team responsible for managing,processing, and sharing massive amounts of data. Accordingly, avalidated pipeline for analyzing paired whole-genome and transcriptomenext-generation sequencing data, such as a Next-Generation DataProcessing and Analysis Pipeline, which utilizes validated softwaretools and produces standard platform-independent formats (FIG. 6), isused. At each step within the pipeline, statistics files are created toensure that all processes have been completed and the files areuncorrupted. Statistics files contain “aligned bases,” “mismatch rate,”and “md5sums.” Early within a pipeline, several additional checks areinvoked, including estimates of overall library coverage, libraryconsistency, quality of bases, and contamination checks. This pipelineis built around (1) standardized file formats, (2) multiple qualitycontrol checks, (3) automated processing, (4) scheduled releases ofsequence data, sequencing alignments, and variant calls, and (5)centralized primary data processing.

Reads are subjected to a series of quality control steps for quantifyingbiases at any given base, and then are parsed into independent FASTQfiles for alignment using BWA-MEM for accurate split-read alignment ofthe unique CDR3 structures. Each sequence has a 12 nucleotide match toone of the V_(κ)or V_(H) gene segments, corresponding to the CASSconsensus amino acid sequence from the second conserved cysteine at the3′ end of the V segment, as well as a 6 nucleotide match to the Jsegment corresponding to the conserved phenylalanine. The total numberof nucleotides between these codons determines the length and thereforethe reading frame of the CDR3 region. Processed sequence data will bedeposited in the ASU secure relational database management system, whichallows a WebApp front end through JasperSoft Server as well as a secureMongoDB instance allowing Ad Hoc querying. Pairing of IgH and IgIosequences form individual cells will be conducted by a basic “if-then”algorithm, searching for complementary base pairing at the 10-merbarcode sequence stretch of each read.

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1. A method for incorporating single-stranded DNA (ssDNA) barcodedpolynucleotides into a nucleic acid origami nanostructure, the methodcomprising: (a) performing first strand synthesis or in-emulsion dropletamplification of a barcode nucleic acid to generate double-strandedbarcode nucleic acids, wherein the barcode nucleic acid comprisesregions complementary to conserved regions of gene sequences of interestas well as at least one priming site complementary to one side of asingle-stranded 5′-5′ bowtie linker nucleic acid comprising a central5′-5′ phosphodiester linker flanked on one side by a sequencecomplementary to a nucleic acid origami nanostructure; (b) combining thedouble-stranded barcode nucleic acids and the 5′-5′ bowtie linkernucleic acid in an oil-emulsion droplet comprising reagents forelongating a target nucleic acid; (c) thermal cycling the oil-emulsiondroplet comprising the double-stranded barcode nucleic acids and the5′-5′ bowtie linker nucleic acid sufficient to result in annealing ofthe double-stranded barcode nucleic acids to complementary sequences onthe 5′-5′ bowtie linker nucleic acid and elongation of ssDNA barcoded5′-5′ bowtie polynucleotides thereby yielding an elongation droplet; (d)extracting elongation products from the elongation droplet; (e)purifying ssDNA barcoded 5′-5′ bowtie polynucleotides from the extractedelongation products; and (f) incorporating the purified ssDNA barcoded5′-5′ bowtie polynucleotides into a nucleic acid origami nanostructure.2. The method of claim 1, wherein incorporating comprises annealing thepurified ssDNA barcoded 5′-5′ bowtie polynucleotides to a nucleic acidorigami nanostructure.
 3. The method of claim 1, wherein the nucleicacid origami nanostructure is a DNA nanostructure.
 4. A method fordetecting target nucleic acid sequences at the single cell level, themethod comprising (a) contacting a nucleic acid origami nanostructureobtained according to the method of claim 1 to nucleic acids within asingle cell, wherein the nanostructure comprises single-strandedbarcoded polynucleotides having capture sequences complementary totarget nucleic acid sequences; and wherein contacting occurs underconditions suitable for binding of the capture sequences to the targetnucleic acid sequences if present in the single cell; (b) recoveringtarget nucleic acid sequences bound to the single-stranded, barcodedpolynucleotide capture sequences; and (c) reverse transcribing therecovered target nucleic acid sequences using the single-strandedbarcoded polynucleotides as gene-specific primers for reversetranscription, whereby target nucleic acids, if present in the cell, aredetected without a single-cell sorting step by sequencing a product ofreverse transcription.
 5. The method of claim 4, wherein the nucleicacid origami nanostructure is a DNA nanostructure.
 6. A method fordetecting B cell receptor sequences at the single cell level, the methodcomprising (a) transfecting into a B cell a nucleic acid origaminanostructure obtained according to the method of claim 1 and comprisingone or more single-stranded barcoded nucleic acid capture sequencescomplementary to immunoglobulin heavy (IgH) and light (IgL) chain B cellreceptor mRNA; (b) contacting the nucleic acid origami nanostructurecomprising one or more single-stranded barcoded nucleic acid capturesequences with immunoglobulin heavy (IgH) and light (IgL) chain B cellreceptor mRNA to bind and protect said mRNA in the transfected B cells;(c) isolating the contacted nucleic acid origami nanostructures torecover IgH and IgL mRNA bound to the one or more single-strandedbarcoded nucleic acid capture sequences; and (d) reverse transcribingthe recovered IgH and IgL mRNA using the one or more single-strandedbarcoded nucleic acid capture sequences as gene-specific primers forreverse transcription, whereby target B cell receptor sequences aredetected without a single-cell sorting step by sequencing a product ofreverse transcription.
 7. The method of claim 6, wherein the nucleicacid origami nanostructures comprise integral biotin labels and whereinisolating the contacted nanostructures comprises avidin columnpurification.
 8. The method of claim 4, wherein said target nucleic acidsequences comprise sequences encoding a cell receptor.
 9. The method ofclaim 8, wherein said sequences encoding a cell receptor comprise B cellreceptor sequences.
 10. The method of claim 4, wherein said sequencesencoding a cell receptor comprise T cell receptor sequences.
 11. Themethod of claim 4, wherein said target nucleic acid sequences comprisenaturally occurring or genomic DNA, RNA, or a synthetic nucleic acidsequence.
 12. The method of claim 4, wherein the nucleic acid origaminanostructures comprise integral biotin labels, wherein the targetnucleic acid sequences are recovered by isolating the contactednanostructures, and wherein isolating the contacted nanostructurescomprises avidin column purification.